Pichia ciferrii cells and use thereof

ABSTRACT

The invention relates to genetically modified  Pichia ciferrii  cells, to the use thereof and to a method of producing sphingoid bases and sphingolipids.

FIELD OF THE INVENTION

The invention relates to genetically modified Pichia ciferrii cells, tothe use thereof and to a method of producing sphingoid bases andsphingolipids.

PRIOR ART

Since the beginning of the 1960s, Pichia ciferrii has been used in theproduction of sphingoid bases and sphingolipids, cf. Wickerham et al.1960, J Bacteriol. 80, 484-91.

It is always worth improving the wild-type strain yields of sphingoidbases and sphingolipids.

It was the object of the invention to make available Pichia ciferriicells which have increased productivity regarding sphingoid bases andsphingolipids.

DESCRIPTION OF THE INVENTION

Surprisingly, we found that the cells described hereinbelow havingreduced, specific enzyme activities are capable of achieving the objectof the invention.

The present invention therefore describes genetically modified Pichiaciferrii cells having, in comparison with their wild type, reducedactivities of the enzymes as described in the present Claim 1.

The invention further relates to the use of the cells mentioned aboveand to a method of producing sphingoid bases and sphingolipids.

One advantage of the present invention is that of the cells according tothe invention being able to grow to high cell densities.

Another advantage of the present invention is that of the cellsproducing markedly increased titres of acetylated sphingoid bases whengrown in appropriate nutrient media.

A further advantage of the present invention is the high geneticstability of the strains which rules out reversion to the originalgenotype. Said high genetic stability moreover allows culturing in theabsence of antibiotics, since there is no selection pressure to bemaintained.

A further advantage of the present invention is the possibility ofemploying the cells in the biotechnological, environmentally friendlyproduction of sphingoid bases from inexpensive and renewable rawmaterials.

The present invention relates to a Pichia ciferrii cell which ischaracterized in that the cell has, compared to its wild type, a reducedactivity of at least one of the enzymes which are encoded by theintron-free nucleic acid sequences selected from the two groups A) andB) consisting of

A) Seq ID No 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9, SeqID No 11,

B) a sequence which is at least 80%, particularly preferably at least90%, additionally preferably at least 95%, and most preferably at least99%, identical to any of the sequences Seq ID No 1, Seq ID No 3, Seq IDNo 5, Seq ID No 7, Seq ID No 9, Seq ID No 11.

In this context, group A) is the nucleic acid sequence group preferredaccording to the invention.

A “wild type” of a cell means in the context of the present inventionpreferably the parent strain from which the cell according to theinvention has evolved through manipulation of the elements (for examplethe genes comprising the specified nucleic acid sequences coding forcorresponding enzymes or the promoters present in corresponding genesand functionally linked to the nucleic acid sequences specified) thatinfluence the activities of the enzymes encoded by the nucleic acid SeqID No specified.

The term “activity of an enzyme” in connection with the enzyme encodedby Seq ID No 1 or 3 or by a sequence at least 80%, particularlypreferably at least 90%, additionally preferably at least 95%, and mostpreferably at least 99%, identical to Seq ID No 1 or 3 is alwaysunderstood as meaning the enzymic activity which catalyses the reaction5,10-methylenetetrahydrofolate+L-glycine+H₂O<=>tetrahydrofolate+L-serine.This activity is preferably determined by the method described inSchlüpen, 2003.

The term “activity of an enzyme” in connection with the enzyme encodedby Seq ID No 5 or by a sequence at least 80%, particularly preferably atleast 90%, additionally preferably at least 95%, and most preferably atleast 99%, identical to Seq ID No 5 is always understood as meaning theenzymic activity which catalyses the reaction L-serine<=>pyruvate+NH₃.

This activity is preferably determined by the method described in Ramosand Wiame, Eur J Biochem. 1982 Apr; 123(3):571-6.

The term “activity of an enzyme” in connection with the enzyme encodedby Seq ID No 7 or by a sequence at least 80%, particularly preferably atleast 90%, additionally preferably at least 95%, and most preferably atleast 99%, identical to Seq ID No 7 is always understood as meaning theenzymic activity which catalyses the reactionATP+sphinganine<=>ADP+sphinganine 1-phosphate.

This activity is preferably determined by the method described inLanterman and Saba, Biochem J. 1998 Jun. 1; 332 (Pt 2):525-31.

The term “activity of an enzyme” in connection with the enzyme encodedby Seq ID No 9 or by a sequence at least 80%, particularly preferably atleast 90%, additionally preferably at least 95%, and most preferably atleast 99%, identical to Seq ID No 9 is always understood as meaning theenzymic activity which catalyses the reaction sphinganine1-phosphate<=>phosphoethanolamine+palmitaldehyde.

This activity is preferably determined by the method described in VanVeldhoven and Mannaerts, J Biol Chem. 1991 Jul. 5; 266(19):12502-7.

The term “activity of an enzyme” in connection with the enzyme encodedby Seq ID No 11 or by a sequence at least 80%, particularly preferablyat least 90%, additionally preferably at least 95%, and most preferablyat least 99%, identical to Seq ID No 11 is understood as meaning thelevel of the rate of expression of the enzyme in question, in particularthe intracellular concentration. This is determined by 2-D geltechnology or Western-blot methods described below.

The wording “reduced activity compared to its wild type” meanspreferably an activity reduced by at least 50%, particularly preferablyby at least 90%, additionally preferably by at least 99.9%, additionallyeven more preferably by at least 99.99% and most preferably by at least99.999%, based on the wild-type activity.

Reduction of the particular activities of the cell according to theinvention compared to its wild type is determined by above-describedmethods of determining the activity by employing, where possible, equalcell numbers/concentrations, the cells having been grown unter identicalconditions such as medium, gassing, agitation, for example. “Nucleotideidentity” in relation to the sequences stated may be determined with theaid of known methods. In general, special computer programs withalgorithms are used which take into account special requirements.

Preferred methods of determining identity firstly generate the highestagreement between the sequences to be compared. Computer programs fordetermining identity include but are not limited to the GCG programpackage including

GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387,Genetics Computer Group University of Wisconsin, Medicine (Wi), and

BLASTP, BLASTN and FASTA (Altschul, S. et al., Journal of MolecularBiology 215 (1990), pages 403-410. The BLAST program may be obtainedfrom the National Center For Biotechnology Information (NCBI) and fromother sources (BLAST manual, Altschul S. et al., NCBI NLM NIH BethesdaND 22894; Altschul S. et al., supra). The known Smith-Waterman algorithmmay also be used for determining nucleotide identity.

Preferred parameters for determining “nucleotide identity” when usingthe BLASTN program (Altschul, S. et al., Journal of Molecular Biology215 (1990), pages 403-410) are:

Expect Threshold: 10 Word size: 28 Match Score: 1 Mismatch Score: −2 Gapcosts: linear

The above parameters are the default parameters in nucleotide sequencecomparison.

The GAP program can also be used with the above parameters.

An identity of 80% according to the above algorithm means in the contextof the present invention 80% identity. The same applies to higheridentities.

The term “which are encoded by the intron-free nucleic acid sequences”clearly sets out that a sequence comparison involving the sequencesstated herein requires the nucleic acid sequences to be compared to becleared of any introns beforehand.

All percentages (%) are percentages by mass, unless stated otherwise.

Cells preferred according to the invention are characterized in thatreduction of the enzymic activity is achieved by modifying at least onegene comprising any of the sequences selected from the nucleic acidsequence groups A) and B) specified hereinabove, the modification beingselected from the group comprising, preferably consisting of, insertionof foreign DNA into the gene, deletion of at least parts of the gene,point mutations in the gene sequence, and exposing the gene to theinfluence of RNA interference, or replacement of parts of the gene withforeign DNA, in particular of the promoter region.

Foreign DNA is understood in this connection as meaning any DNA sequencewhich is “foreign” to the gene (and not to the organism), i.e. evenPichia ciferrii endogenous DNA sequences may act as “foreign DNA” inthis connection.

In this context, particular preference is given to the gene beingdisrupted by insertion of a selection marker gene, thus the foreign DNAbeing a selection marker gene, in particular one comprising a sequencecoding for the Streptomyces noursei nat1 gene, which sequence ispreferably flanked by the sequence of the Pichia PDA1 promoter and thesequence of the Pichia TEF terminator, as described in Schorsch et al.,2009; Current Genetics (2009), 55(4), 381-389, for example, the sequencecoding for the Streptomyces noursei nat1 gene preferably beingcodon-optimized for P. ciferrii, with said insertion preferably havingbeen accomplished by homologous recombination into the gene locus.

In this connection, it may be advantageous for the selection marker geneto be extended by further functionalities which in turn make subsequentremoval from the gene possible, which can be achieved, for example, byrecombination systems foreign to the organism, such as a Cre/IoxP systemor FRT (flippase recognition target) system, or the organism's ownhomologous recombination system.

Preference is given according to the invention to the cell having,compared to its wild type, a combination of reduced activities of theenzymes which are encoded by the intron-free nucleic acid sequences:

Seq ID No 1 or its group B analogue;

Seq ID No 3 or its group B analogue;

Seq ID No 5 or its group B analogue;

Seq ID No 7 or its group B analogue;

Seq ID No 9 or its group B analogue;

Seq ID No 11 or its group B analogue;

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue;

Seq ID No 1 or its group B analogue and Seq ID No 5 or its group Banalogue;

Seq ID No 3 or its group B analogue and Seq ID No 5 or its group Banalogue;

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue and Seq ID No 5 or its group B analogue;

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or itsgroup B analogue;

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue and Seq ID No 5 or its group B analogue and Seq ID No 11 or itsgroup B analogue;

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or itsgroup B analogue and Seq ID No 11 or its group B analogue;

In connection with the combinations listed above, preference is given toreducing the enzyme activities encoded by the members of group A.

Cells preferred according to the invention are characterized in that thePichia ciferrii cell derives from strains selected from the groupconsisting of Pichia ciferrii NRRL Y-1031 F-60-10, the Pichia ciferriistrains disclosed in the examples of WO 95/12683, and the strain Pichiaciferri CS.PCΔPro2, described in Schorsch et al., 2009, Curr Genet. 55,381-9.

Cells preferred according to the invention are characterized in that thecell has, compared to its wild type, an increased enzymic activity of atleast one of the enzymes selected from

an enzyme E₁, which catalyses the reaction of serine and palmitoyl-CoAto give 3-ketosphinganine, in particular a serine palmitoyl transferase,in particular those encoded by Seq ID No 13 and/or Seq ID No 15,

an enzyme E₂, which catalyses the reaction of sphinganine tophytosphingosine, in particular a sphinganine C4-hydroxylase, inparticular that encoded by Seq ID No 17.

The term “activity of an enzyme” in connection with the enzyme E₁ isalways understood as meaning the enzymic activity which catalyses thereactions of palmitoyl-CoA+L-serine<=>CoA+3-dehydro-D-sphinganine+CO₂.

This activity is preferably determined by the method described inZweerink et al., J Biol Chem. 1992 Dec. 15; 267(35):25032-8.

The term “activity of an enzyme” in connection with the enzyme E₂ isalways understood as meaning the enzymic activity which catalyses thereaction sphinganine+NADPH+H⁺+O₂<=>phytosphingosine+NADP⁺+H₂O.

This activity is preferably determined by the method described inGrilley et al., J Biol Chem. 1998 May 1; 273(18):11062-8.

The term “increased activity of an enzyme” as used hereinabove and inthe comments below in the context of the present invention is preferablyunderstood as meaning increased intracellular activity.

The following comments regarding the increase in enzyme activity incells apply both to the increase in activity of the enzymes E₁ to ₂ andto all enzymes specified hereinbelow, whose activity may be increasedwhere appropriate.

In principle, an increase in enzymic activity can be achieved byincreasing the copy number of the gene sequence(s) coding for theenzyme, by using a strong promoter, by altering the codon usage of thegene, by increasing in various ways the half life of the mRNA or of theenzyme, by modifying the regulation of expression of the gene, or byutilizing a gene or allele coding for a corresponding enzyme withincreased activity, and by combining these measures where appropriate.Cells genetically modified according to the invention are generated, forexample, by transformation, transduction, conjugation or a combinationof these methods with a vector containing the desired gene, an allele ofthis gene or parts thereof and a promoter enabling the gene to beexpressed. Heterologous expression is achieved in particular byintegrating the gene or alleles into the chromosome of the cell or anextrachromosomally replicating vector. An overview of the options forincreasing enzyme activity in cells is given for pyruvate carboxylase byway of example in DE-A-100 31 999 which is hereby incorporated by way ofreference and whose disclosure forms part of the disclosure of thepresent invention regarding the options for increasing enzyme activityin cells.

Expression of the enzymes or genes specified hereinabove and all enzymesor genes specified below is detectable with the aid of one- andtwo-dimensional protein gel fractionation and subsequent opticalidentification of protein concentration in the gel using appropriateevaluation software.

If the increase in an enzyme activity is based exclusively on anincrease in expression of the corresponding gene, the increase in saidenzyme activity can be quantified simply by comparing the one- ortwo-dimensional protein fractionations of wild-type and geneticallymodified cells. A customary method of preparing the protein gels in thecase of bacteria and of identifying the proteins is the proceduredescribed by Hermann et al. (Electrophoresis, 22: 1712.23 (2001). Theprotein concentration may likewise be analysed by Western-blothybridization with an antibody specific to the protein to be detected(Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) andsubsequent optical evaluation using appropriate software forconcentration determination (Lohaus and Meyer (1989) Biospektrum, 5:32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647).

Preference is given according to the invention to the cell which,compared to its wild type, has an increased enzymic activity of enzymeE₁ having, in comparison with its wild type, a combination of reducedactivities of the enzymes encoded by the intron-free nucleic acidsequences:

in the combination of Seq ID No 1 or its group B analogue and Seq ID No3 or its group B analogue and Seq ID No 5 or its group B analogue,

or in the combination of Seq ID No 1 or its group B analogue and Seq IDNo 3 or its group B analogue and Seq ID No 5 or its group B analogue andSeq ID No 7 or its group B analogue and Seq ID No 11 or its group Banalogue.

Preference is given according to the invention to the cell which,compared to its wild type, has an increased enzymic activity of enzymesE₁ and E₂ having, in comparison with its wild type, a combination ofreduced activities of the enzymes encoded by the intron-free nucleicacid sequences:

Seq ID No 1 or its group B analogue and Seq ID No 3 or its group Banalogue and Seq ID No 5 or its group B analogue and Seq ID No 7 or itsgroup B analogue and Seq ID No 11 or its group B analogue.

In one variant embodiment, P. ciferrii cells according to the inventionare such as those described in WO2006048458 and WO2007131720 andadditionally having the changes in enzymic activities described above inconnection with the present cells according to the invention.

A further contribution to achieving the object of the invention is madeby using the cells according to the invention for producing sphingoidbases and sphingolipids.

The term “sphingoid bases” in the context of the present invention isunderstood as meaning phytosphingosine, sphingosine, sphingadienine,6-hydroxysphingosine and sphinganine (dihydrosphingosine), also in theacetylated form, such as for example tetraacetylphytosphingosine,triacetylphytosphingosine, diacetylphytosphingosine,O-acetylphytosphingosine, triacetylsphinganine, diacetylsphinganine,O-acetylsphinganine, triacetylsphingosine, diacetylsphingosine,O-acetylsphingosine, tetraacetyl-6-hydroxysphingosine,triacetyl-6-hydroxysphingosine, diacetyl-6-hydroxysphingosine,O-acetyl-6-hydroxysphingosine, triacetylsphingadienine,diacetylsphingadienine, O-acetylsphingadienine.

The term “sphingolipids” in the context of the present invention isunderstood as meaning compounds which comprise sphingoid basescovalently linked via an amide bond to a fatty acid. The fatty acid maybe saturated or mono- or polyunsaturated. The fatty acid side chain mayvary in length. The fatty acid side chain may also have functionalgroups such as hydroxy groups. Sphingolipids include, for example,phytoceramides, ceramides and dihydroceramides, and the more complexglucosylceramides (cerebrosides) and the inositol phosphorylceramides,mannosylinositol phosphorylceramides and mannosyldiinositolphosphorylceramides. The sphingolipids here also include sphingoid baseslinked via an amide bond to an acetyl radical, such as for exampleN-acetylphytosphingosine, N-acetylsphinganine, N-acetylsphingosine,N-acetyl-6-hydroxysphingosine. These compounds are also known by theterm of short-chain ceramides.

The use of the cells according to the invention for producing sphingoidbases and sphingolipids selected from the group consisting ofphytosphingosine, sphingosine, sphingadienine, 6-hydroxysphingosine,sphinganine (dihydrosphingosine), tetraacetylphytosphingosine (TAPS),triacetylphytosphingosine, diacetylphytosphingosine,O-acetylphytosphingosine, N-acetylphytosphingosine, triacetylsphinganine(TriASa), diacetyisphinganine, O-acetylsphinganine, N-acetylsphinganine,triacetylsphingosine (TriASo), diacetylsphingosine, O-acetylsphingosine,N-acetylsphingosine, tetraacetyl-6-hydroxysphingosine,triacetyl-6-hydroxysphingosine, diacetyl-6-hydroxysphingosine,O-acetyl-6-hydroxysphingosine, N-acetyl-6-hydroxysphingosine,triacetylsphingadienine, diacetylsphingadienine, O-acetylsphingadienineis particularly advantageous. Very particular preference is given to theuse of the cells according to the invention for producingtetraacetylphytosphingosine (TAPS).

A use which is preferred according to the invention is characterizedaccording to the invention in that cells preferred according to theinvention, as described above, are used.

P. ciferrii cells which are used in particular for producing theabove-described sphingosine and sphinganine derivatives are such asthose described in WO2006048458 and WO2007131720 and additionally havingthe changes in enzymic activities described above in connection with thepresent cells according to the invention.

A further contribution to achieving the object of the invention is madeby a method of producing the previously described cell according to theinvention, said method comprising the steps of:

I) providing a Pichia ciferrii cell, and

II) modifying at least one gene comprising any of the sequences selectedfrom the nucleic acid sequence groups A) and B) specified in Claim 1 byinsertion of foreign DNA, in particular DNA coding for a selectionmarker gene, preferably one which can be removed without leaving a traceand which leaves a deletion in the target gene, into the gene, deletionof at least parts of the gene, point mutations in the gene sequence,exposing the gene to the influence of RNA interference, and replacementof parts of the gene with foreign DNA, in particular of the promoterregion.

A further contribution to achieving the object of the invention is madeby a method of producing sphingoid bases and sphingolipids, said methodcomprising the steps of

a) contacting the cell according to the invention with a mediumincluding a carbon source,

b) culturing the cell under conditions which enable the cell to producesphingoid bases and sphingolipids from said carbon source, and

c) optionally isolating the sphingoid bases and sphingolipids produced.

Methods preferred according to the invention employ cells specifiedabove as preferred according to the invention.

Carbon sources which may be employed are carbohydrates, such as forexample glucose, fructose, glycerol, sucrose, maltose, molasses, or elsealcohols, such as for example ethanol, and organic acids, such as forexample acetate. Nitrogen sources which may be employed are for exampleammonia, ammonium sulphate, ammonium nitrate, ammonium chloride, organicnitrogen compounds (such as yeast extract, malt extract, peptone, cornsteep liquor). Inorganic compounds, such as for example phosphate salts,magnesium salts, potassium salts, zinc salts, iron salts and others, mayalso be employed.

Suitable culturing conditions for Pichia ciferri are known to theskilled worker from WO2006048458 and WO2007131720, for example.

The method according to the invention is particularly suitable forproducing tetraacetylphytosphingosines (TAPS).

The Examples listed hereinbelow describe the present invention by way ofexample, but the embodiments specified in said examples are not intendedto limit the invention, the scope of use of which ensues from the entiredescription and the claims.

The following figures are part of the Examples:

FIG. 1: Design principle of gene deletion cassettes

FIG. 2: Design principle of overexpression cassettes

EXAMPLES Construction of Gene Deletion Cassettes

Unless stated otherwise, gene deletions were carried out by means ofclassical “one-step gene replacement”, as described in Rothstein 1983,Methods Enzymol 101: 202-211.

Deletion cassettes were constructed by in vivo cloning, ultimatelyresulting in plasmids which were used as templates for PCR-basedamplification of the deletion cassettes. These PCR products were thentransformed into P. ciferrii with the aim of deleting a particular gene.

The deletion cassettes were constructed by employing the plasmidp426HXT7-6HIS (Hamacher et al., 2000; Microbiology 148, 2783-8) asshuttle vector. p426HXT7-6HIS was first cleaved with BamHI and EcoRI,resulting in a 5.69 kb fragment which was used as backbone for thesubsequent cloning steps. Initially, three overlapping DNA fragmentswere generated by PCR for each P. ciferrii deletion cassette: a dominantclonNAT marker, which could later be eliminated again, as the centralpart (nat1 resistance cassette) (cf. Schorsch et al., Curr Genet. 2009Aug; 55(4):381-9), a second fragment of about 500 by in length,representing the 5′-untranslated region of the ORF to be deleted(promoter region, PR) and with overlap to the start of theclonNAT-marker fragment, and a third fragment of about 500 by in length,representing the 3′-untranslated region (terminator region, TR) of theORF to be deleted and with overlap to the end of the clonNAT-markerfragment.

Each deletion cassette was constructed by amplifying by means of PCR thepromoter region (PR) and the terminator region (TR) of the gene to bedeleted from genomic P. ciferrii wild-type DNA, in each case employinggene-specific primers. To this end, primer pairs, P1/P2 for PR and P3/P4for TR, were used in each case. The primers were chosen so as to have atthe 5′ end regions of about 30-35 bps in length which were overlappingwith the DNA elements to be fused:

Primer 5′ End overlapping with: P1 Cloning vector p426HXT7-6HIS P2 nat1Resistance cassette (PCR amplicon of plasmid pCS.LoxP.nat1 with primersLPNTL.fw and LPNTL.rv) P3 nat1 Resistance cassette P4 Cloning vectorp426HXT7-6HIS

The central fragment (nat1 resistance cassette, Seq ID No 19) wasamplified using in each case the primer pair LPNTL.fw(TGGCGCTTCGTACCACTGGGTAAC) and LPNTL.rv (GAAATTAATACGACTCACTATAGG), withplasmid pCS.LoxP.nat1 (Schorsch et al., 2009; Curr. Genet. 55, 381-9)being employed as template (all primer sequences are given in 5′→3′orientation).

The PCR products of primer pairs P1/P2, P3/P4 and LPNTL.fw/LPNTL.rv,together with the p426HXT7-6HIS plasmid previously linearized bydigestion with BamHI and EcoRI, were transformed into S. cerevisiaestrain K26. The PCR products and the linearized vector were joinedtogether in vivo by homologous recombination, causing the linearizedvector to be re-circularized and able to be propagated in S. cerevisiae.Transformants obtained were selected by means of the marker gene (nat1)on YEPD plates with clonNAT, their DNA was isolated and transformed intoE. coli, and the plasmids re-isolated therefrom were verified byrestriction mapping or sequencing. The deletion cassettes were amplifiedusing the primer pairs 426L.fw (GCTTCCGGCTCCTATGTTG, Seq ID No 23) and426R.rv (ACCCTATGCGGTGTGAAATAC, Seq ID No 24) or HXT7(GCCAATACTTCACAATGTTCGAATC, Seq ID No 25) and CYC(CGTGAATGTAAGCGTGACATAAC, Seq ID No 26), unless stated otherwise. SeeFIG. 1 for clarification.

To successively delete multiple genes, a marker rescue was performedafter each deletion. This was accomplished by transformation withplasmid pCS.opt.Cre (Seq ID. No 20) as described previously (Schorsch etal., Curr Genet. 2009 Aug; 55(4):381-9). The gene deletions wereverified by PCR analyses using genomic DNA of the transformants astemplate.

The particular gene deletion cassettes of the genes with sequences SeqID No 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9 and Seq IDNo 11 were constructed using the primers listed in the table below. Foreach of the Seq IDs, the first two primers listed (SH11 and SH12 or SH21and SH22 or C1 and C2 or HXT7-LCB4.fw and LCB4.HXT7.rv or HXT7-DPL1.fwand DPL1.rv2 or ORM-426L.fw and ORM-LPNTL.rv) were used in each case foramplification of PR, with the next two primers listed (SH13 and SH14 orSH23 and SH24 or C3 and C4 or LCB4.rv and LCB4.fw or DPL1.fw2 andCYC-DPL1.rv or ORM-LPNTL.fw2 and ORM-426R.rv) being used foramplification of TR. The last two primers listed in each case(SHMT1.pop-in.fw and SHMT1.veri.rv or SHMT2.pop-in.fw and SHMT2.veri.rvor CHA1.pop-in.fw and CHA1.veri.rv or LCB4.pop-in.fw and LCB4.veri.rv orDPL1.pop-in.fw and DPL1.veri.rv or ORM1.pop-in.fw and ORM.veri.rv) areused for detecting integration or the wild-type allele.

Gene Primer name Sequence (5′->3′) Seq ID No 1 SH11CAAAAAGTTAACATGCATCACCATCACCATCACACT Seq ID No 27 AACCCAACTAGGCTCATTAACSH12 GTTATCTGCAGGTTACCCAGTGGTACGAAGCGCCA Seq ID No 28TCAGCCATTTCTGGATCAATTTC SH13 TGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCATSeq ID No 29 CCAGTTCCAGGTGAATTATAAG SH14TAACTAATTACATGACTCGAGGTCGACGGTATCCCA Seq ID No 30 TACTATGCTTGGCATCTTAAACSHMT1.pop-in.fw TTGATAGGGCAAATTCTCCAAC Seq ID No 31 SHMT1.veri.rvTTCACCTGGATAACCTTCTG Seq ID No 32 Seq ID No 3 SH21CAAAAAGTTAACATGCATCACCATCACCATCACATG Seq ID No 33 TCCTTGCAGGTGGTATTCSH22 TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCAG Seq ID No 34GTAAAGCGTATGGCATGTTG SH23 CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGSeq ID No 35 CTGGTGAATTCCCATTATCTG SH24TAACTAATTACATGACTCGAGGTCGACGGTATCCAT Seq ID No 36 AACCATCTAAAGCATTATAGTCSHMT2.pop-in.fw AAGTTTCAGCAAATGGTTTGAC Seq ID No 37 SHMT2.veri.rvTATCTTGCACCTGGATAACC Seq ID No 38 Seq ID No 5 C1CAAAAAGTTAACATGCATCACCATCACCATCACAAT Seq ID No 39CTAAGAGGTAAAGTTCAACATTC C2 GTTATCTGCAGGTTACCCAGTGGTACGAAGCGCCASeq ID No 40 TTGGTTTGCCGTGTGGATTG C3CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCG Seq ID No 41  GAGTTCAACAACCGTTCAAGC4 TAACTAATTACATGACTCGAGGTCGACGGTATCATG Seq ID No 42 AAGTTGATGCTGCTTTGGCHA1.pop-in.fw ATTTAGAAGCTAGAGGTTCAGAAAG Seq ID No 43 CHA1.veri.rvTAGAAGAATGACCATGCCATATAG Seq ID No 44 Seq ID No 7 HXT7-LCB4.fwTTTTAATTTTAATCAAAAAGTTAACATGCATCACCAT Seq ID No 45CACCATCACACTCACAGAGTCAACTCCTGTATATTC LCB4.HXT7.rvTGAATGTAAGCGTGACATAACTAATTACATGACTCG Seq ID No 46AGGTCGACGGTATCTCTGGCGGTATTGAACTTTGT GGAG LCB4. rvGTTATCTGCAGGTTACCCAGTGGTAAAGTGTATGGA Seq ID No 47TGGGTTGAAGTATGTCTTTATATC LCB4.fw ACGAAGTTATGAGCTCGAATTCATCGATGCTACCCGSeq ID No 48 GTGCTGCAAAGACTTTACTAAG LCB4.pop-in.fwGTGAATGGTTAATAGTGCGCTATG Seq ID No 49 LCB4.veri.rvCTAACAAATACCACTTCGACATCAG Seq ID No 50 Seq ID No 9 HXT7-DPL1.fwTTTTAATTTTAATCAAAAAGTTAACATGCATCACCAT Seq ID No 51CACCATCACACCTTCCGTGAGATTTCCCTTGTTTAC DPL1.rv2TATACGAAGTTATCTGCAGGTTACCCAGTGGTATAA Seq ID No 52 CCCATAACCAGTGATGTTAACCDPL1.fw2 GAAGTTATGAGCTCGAATTCATCGATGACCACTGGT Seq ID No 53 GTTGTTGATCGCYC-DPL1.rv TGAATGTAAGCGTGACATAACTAATTACATGACTCG Seq ID No 54AGGTCGACGGTATCCGACGGTAATGAGGATGTAAA TGAG DPL1.pop-in.fwAAACAAGAGCAGCATGCAACTTGAG Seq ID No 55 DPL1.veri.rvAGTGACACCAGGAACTCTAAAG Seq ID No 56 Seq ID No 11 ORM-426L.fwGCTTTACACTTTATGCTTCCGGCTCCTATGTTGAAC Seq ID No 57 TATGTCAATATCGATCGTATGORM-LPNTL.rv TATCTGCAGGTTACCCAGTGGTACGAAGCGCCAAA Seq ID No 58CAGAAATTGGTTCATGTGTTG ORM-LPNTL.fw2 GCCGGTCTCCCTATAGTGAGTCGTATTAATTTCTGGSeq ID No 59 TGTACCAATTTGGTTATTTC ORM-426R.rvATATCAGTTATTACCCTATGCGGTGTGAAATACACA Seq ID No 60 AGTACAACAACAACAGATTTAGORM1.pop-in.fw TACCCACCTTTGACATAATCAG Seq ID No 61 ORM.veri.rvATTCAAATGGCGTACCTTTAAC Seq ID No 62

Construction of Overexpression Cassettes

Overexpression cassettes were constructed in principle in the same wayas the method used for the deletion cassettes. In the case ofoverexpression cassettes, however, an additional fourth PCR product wasgenerated (promoter fragment, PF), representing a fragment of the PcTDH3or the PcENO1 promoter. This was later linked in vivo to the nat1resistance cassette and the third PCR fragment which in this case had anoverlap with the start of the ORF to be overexpressed (see FIG. 2).

For overexpression of the gene product Seq ID No 13, the native promoterin P. ciferrii was replaced with the PcENO1⁻⁵⁸⁴⁻¹ (Seq ID No 21)promoter fragment. In contrast, the particular native promoter in P.ciferrii was replaced with the PcTDH3⁻⁴²⁰⁻¹ promoter fragment (Seq ID No22) for overexpression of the gene products Seq ID No 15 and Seq ID No17.

In principle, three different gene-specific primer pairs were used forconstructing the particular overexpression cassettes. The primers werechosen so as to have at the 5′ end regions of about 30-35 bps in lengthwhich were overlapping with the DNA elements to be fused.

Primer 5′ End overlapping with: P5 Cloning vector p426HXT7-6HIS P6 nat1Resistance cassette (PCR amplicon of plasmid pCS.LoxP.nat1 with primersLPNTL.fw and LPNTL.rv) P9 nat1 Resistance cassette P10 5′-End of the ORFto be overexpressed P7 3′-End of the PcENO1⁻⁵⁸⁴⁻¹ or PcTDH3⁻⁴²⁰⁻¹promoter fragment P8 Cloning vector p426HXT7-6HIS

The nat1 resistance cassette was amplified using in each case the primerpair LPNTL.fw and LPNTL.rv, with plasmid pCS.LoxP.nat1 (Schorsch et al.,2009; Curr. Genet. 55, 381-9) being employed as template. The PCRproducts of primer pairs P5/P6, P7/P8, P9/P10 and LPNTL.fw/LPNTL.rv,together with the p426HXT7-6HIS plasmid previously linearized bydigestion with Hpal and NgoMIV, were transformed into S. cerevisiaestrain K26. The PCR products and the linearized vector were joinedtogether in vivo by homologous recombination, causing the linearizedvector to be re-circularized and able to be propagated in S. cerevisiae.Transformants obtained were selected by means of the marker gene (nat1)on YEPD plates with clonNAT, their DNA was isolated and transformed intoE. coil, and the plasmids re-isolated therefrom were verified byrestriction mapping or sequencing. The overexpression cassettes wereamplified using the primer pair “426L.fw & 426R.rv” in each case.

Cf. FIG. 2 for clarification.

For combined overexpression of multiple genes, or for combiningoverexpressions of one or more target genes with one or more genedeletions, a marker rescue was performed after each step (deletion of atarget gene or chromosomal integration of an overexpression cassette).This was accomplished by transformation with plasmid pCS.opt.Cre asdescribed previously (Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9). Integration of the overexpression cassettes was verifiedby PCR analyses using genomic DNA of the transformants as template.

The particular overexpression cassettes for enzymes encoded by thesequences Seq ID No 13, Seq ID No 15 and Seq ID No 17 were constructedusing the primers listed in the table below. For each of the Seq IDs,the first two primers listed (LCB1.426L.fw and LCB1.LPNTL.rv orLCB2-426L.fw and LCB2-LPNTL.rv or SYR2oe.426L and SYR2oe.LPNTL.rv) wereused in each case for amplification of PR. The next two primers listed(P-ENO.LPNTL.fw and LCB1.P-ENO.rv or TDH3-LPNTL.fw and P-TDH3.rv orTDH3-LPNTL.fw and P-TDH3.rv) were used for amplification of theparticular PcENO1⁻⁵⁸⁴⁻¹ or PcTDH3⁻⁴²⁰⁻¹ promoter fragment. The next twoprimers listed (P-ENO.LCB1.fw and LCB1.426R.rv or LCB2.P-TDH3.fw andLCB2-426R.rv or SYR2oe.P-TDH3.fw and SYR2oe.426R) were used foramplification of the 5′-ORF fragments of the target genes to beoverexpressed in each case. The last two primers listed in each case(P-ENO.veri.rv and LCB1üe.veri.rv or P-TDH3.pop.fw and LCB2üe.veri.rv orP-TDH3.pop.fw and SYR2oe.veri.rv) are used for detecting integration orthe wild-type allele.

Gene Primer name Sequence (5′->3′) Seq ID No 13 LCB1.426L.fwGCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGACTGCT Seq ID No 63 ACACTCCAAATATGLCB1.LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCATAATAG Seq ID No 64AAGAAACACGTCAAATACC P-ENO.LPNTL.fwGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCCAGATCAA Seq ID No 65 ACCACATCATGAGLCB1.P-ENO.rv GTAGCAGTGACGTTCATTGTGTAATGTGTATATGTTTTATC Seq ID No 66P-ENO.LCB1.fw CATATACACATTACACAATGAACGTCACTGCTACAAC Seq ID No 67LCB1.426R.rv ATATCAGTTATTACCCTATGCGGTGTGAAATACACAAGCAC Seq ID No 68CAACACCATTAC P-ENO.veri.rv GTTGTGCGTGGCTTGAC Seq ID No 69 LCB1üe.verisvATAATACAGCACCACCAACTTC Seq ID No 70 Seq ID No 15 LCB2-426L.fwGCTTTACACTTTATGCTTCCGGCTCCTATGTTGGGCCATGA Seq ID No 71 GATGACTTTGTACGLCB2-LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCAGTTCTT Seq ID No 72GTTTGAATTCGCGTTTG TDH3-LPNTL.fwGTTATGAGCTCGAATTCATCGATGATATCAGGGACCGTTAA Seq ID No 73 TTACCAACAATCTCP-TDH3.rv TGTTAATTAATTATTTGTTTGTTTG Seq ID No 74 LCB2.P-TDH3.fwACAAACAAACAAACAAATAATTAATTAACAATGTCATTGGTA Seq ID No 75 ATACCTCAAATAGLCB2-426R.rv ATATCAGTTATTACCCTATGCGGTGTGAAATACAAAGCGGC Seq ID No 76TTGAGTACATGC P-TDH3.pop.fw AACTGACGTTTCAAGAACATC Seq ID No 77LCB2üe.veri.rv ATAAACTTGCATTTGTTGCATACC Seq ID No 78 Seq ID No 17SYR2oe.426L GCTTTACACTTTATGCTTCCGGCTCCTATGTTGAAAGTGTA Seq ID No 79AATAGACGTCATGAG SYR2oe.LPNTL.rv TTATCTGCAGGTTACCCAGTGGTACGAAGCGCCACTGTGTSeq ID No 80 ACTAAACGTGATAAATCC TDH3-LPNTL.fwGTTATGAGCTCGAATTCATCGATGATATCAGGGACCGTTAA Seq ID No 81 TTACCAACAATCTCP-TDH3.rv Seq ID No 82 TGTTAATTAATTATTTGTTTGTTTG SYR2oe.P-TDH3.fwAAAACAAACAAACAAACAAATAATTAATTAACAATGAGCTCT Seq ID No 83 CATCAGTTTTTGSYR2oe.426R ATATCAGTTATTACCCTATGCGGTGTGAAATACAAGACGAT Seq ID No 84GATGTCTTGAATG P-TDH3.pop.fw AACTGACGTTTCAAGAACATC Seq ID No 85SYR2oe.veri.rv AGTAACAATTGCAGCAATACC Seq ID No 86

Production of Acetylated Sphingoid Bases by the Genetically ModifiedStrains

Increased titres of acetylated sphingoid bases were achieved by thefollowing genetic modifications:

The tables below depict the titres of acetylated sphingoid bases(tetraacetylphytosphingosine, TAPS and optionally triacetylsphinganine,TriASa) of the different recombinant P. ciferrii strains after growth tothe stationary phase in a shaker flask.

Details (media used, growth conditions, extraction, quantification byHPLC analysis) are described in Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9. The strain employed in the present application correspondsto Pichia ciferri CS.PCΔPro2 designated in the above reference, which isalso referred to for short as “CS” hereinbelow.

First, the influence of deletions of various genes on the production ofacetylated sphingoid bases was investigated. The results are depicted inthe table below. Individually, deletion of PcSHM2 in particular wasshown to markedly increase production of acetylated sphingoid bases.This effect was further enhanced by the combination with a PcSHM1deletion. Further enhancement was achieved by an additional deletion ofPcCHA1. This strain, with the relevant genotype of cha1 shm1 shm2,yielded by far the highest titre of 64 mg of TAPS*g−1 (CDW) plus 3 mg ofTriASa*g−1 (CDW).

Influence of deletions of various genes on production of acetylatedsphingoid bases:

Relevant mg of TAPS * mg of TriASa * Strain genotype¹ g⁻¹ (CDW) g⁻¹(CDW)² CS 21 CS.S1 shm1 20 CS.S2 shm2 26 CS.SS shm1shm2 42 CS.C cha1 23CS.CS1 cha1 shm1 23 CS.CS2 cha1 shm2 29 CS.CSS cha1 shm1 shm2 65 3¹Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-ID No 3; cha1,SEQ-ID No 5 ²Titres below 2 mg/g of cell dry mass are not shown.

Next, the influence of various genetic modifications for enhancingenzyme activities were investigated, in the background of strain CS.CSS(cha1 shm1 shm2). For this purpose, the following genetic modifications,both individual and by way of selected combinations, were carried out inthe strain CS.CSS:

deletion of PcLCB4, Seq ID No 7

deletion of PcDPL1, Seq ID No 9

deletion of PcORM12, Seq ID No 11

overexpression of PcLCB1 Seq ID No 13

overexpression of PcLCB2 Seq ID No 15

overexpression of PcSYR2 Seq ID No 17.

Moreover, the effects of the deletions of PcLCB4 and PcDPL1 were alsoaddressed alone, that is without combination with the cha1 shm1 shm2genotype.

To achieve additive or synergistic effects, a multiplicity of thegenetic modifications promoting sphingoid base production were combinedin different ways in a single strain. The strain with the followinggenotype turned out to be the best here: cha1 shm1 shm2 lcb4 orm12TDH3p:LCB2 ENO1p:LCB1 TDH3p:SYR2. This strain produced in a shaker flaska titre of 199 mg of TAPS*g⁻¹ (CDW) (plus 12 mg of triacetylsphinganine(TriASa)*g⁻¹ (CDW), while the CS reference strain produced only 21 mg ofTAPS*g⁻¹ (CDW).

The results are depicted in the table below.

Influence of genetic modifications of sphingolipid metabolism onproduction of acetylated sphingoid bases

Relevant mg of TAPS * mg of TriASa * Strain genotype^(1; 2) g⁻¹ (CDW)g⁻¹ (CDW)³ CS 21 CS.L4 lcb4 54 CS.DPL1 dpl1 28 CS.S2 shm2 26 CS.SS shm1shm2 42 CS.CSS cha1 shm1 shm2 65 3 CSS.L1 cha1 shm1 shm2 74 2 NO1p:LCB1CSS.L2 cha1 shm1 shm2 82 4 TDH3p:LCB2 CSS.L1.L2 cha1 shm1 shm2 102 8ENO1p:LCB1 TDH3p:LCB2 CSS.L4 cha1 shm1 shm2 116 8 lcb4 CSS.O cha1 shm1shm2 104 6 orm12 CSS.D cha1 shm1 shm2 84 3 dpl1 CSS.L4.O cha1 shm1 shm2172 8 lcb4 orm12 CSS.L4.O.L2 cha1 shm1 shm2 182 16 lcb4 orm12 TDH3p:LCB2CSS.L4.O.L2.L1 cha1 shm1 shm2 178 44 lcb4 orm12 TDH3p:LCB2 ENO1p:LCB1CSS.L4.O.L2.L1.S2 cha1 shm1 shm2 199 12 lcb4 orm12 TDH3p:LCB2 ENO1p:LCB1TDH3p:SYR2 ¹Relationship with SEQ-IDs: shm1, SEQ-ID No 1; shm2, SEQ-IDNo 3; cha1, SEQ-ID No lcb4, SEQ-ID No 7; dpl1, SEQ-ID No 9; orm12,SEQ-ID No 11; LCB1, SEQ-ID No 13; LCB2, SEQ-ID No 15; SYR2, SEQ-ID No 17² Inactivated genes are listed in lower-case letters. Overexpressedgenes are listed in capital letters and with the particular promoter(abbreviation “p”), under the control of which they are. ³Titres below 2mg/g of cell dry mass are not shown.

1. An isolated Pichia ciferrii cell, characterized in that the cell has,compared to its wild type, a reduced activity of at least one of theenzymes which are encoded by the intron-free nucleic acid sequencesselected from the two groups A) and B) consisting of A) Seq ID No 1, SeqID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9, Seq ID No 11, B) asequence which is at least 80% identical to any of the sequences Seq IDNo 1, Seq ID No 3, Seq ID No 5, Seq ID No 7, Seq ID No 9, Seq ID No 11.2. An isolated Pichia ciferrii cell according to claim 1, characterizedin that reduction of the enzymic activity is achieved by modifying agene comprising any of the nucleic acid sequences specified in claim 1,the modification being selected from the group comprising insertion offoreign DNA into the gene, deletion of at least parts of the gene, pointmutations in the gene sequence, exposing the gene to the influence ofRNA interference, and replacement of parts of the gene with foreign DNA.3. An isolated Pichia ciferrii cell according to claim 2, characterizedin that the foreign DNA is a selection marker gene, preferably one whichcan be removed without leaving a trace and which leaves a deletion inthe target gene.
 4. An isolated Pichia ciferrii cell according to claim1, characterized in that the Pichia ciferrii cell derives from strainsselected from the group consisting of Pichia ciferrii NRRL Y-1031F-60-10, Pichia ciferrii strains disclosed in WO 95/12683 Pichiaciferrii CS.PCΔPro2.
 5. An isolated Pichia ciferrii cell according toclaim 1, characterized in that the cell has, compared to its wild type,an increased enzymic activity of at least one of the enzymes selectedfrom an enzyme E₁, which catalyses the reaction of serine andpalmitoyl-CoA to give 3-ketosphinganine, an enzyme E₂, which catalysesthe reaction of sphinganine to phytosphingosine.
 6. (canceled)
 7. Amethod of producing an isolated Pichia ciferri cell according to claim1, said method comprising the steps of: I) providing an isolated Pichiaciferrii cell, and II) modifying at least one gene comprising any of thesequences selected from the nucleic acid sequence groups A) and B)specified in claim 1 by insertion of foreign DNA, into the gene,deletion of at least parts of the gene, point mutations in the genesequence, exposing the gene to the influence of RNA interference, andreplacement of parts of the gene with foreign DNA.
 8. A method ofproducing sphingoid bases and sphingolipids, said method comprising thesteps of a) contacting the cell according to claim 1 with a mediumincluding a carbon source, b) culturing the cell under conditions whichenable the cell to produce sphingoid bases and sphingolipids from saidcarbon source, and c) optionally isolating the sphingoid bases andsphingolipids produced.
 9. An isolated Pichia ciferrii cell according toclaim 2, wherein said replacement of parts of the gene with foreign DNAis of the promoter region.
 10. An isolated Pichia ciferrii cellaccording to claim 5, wherein said enzyme E₁ is a serine palmitoyltransferase.
 11. An isolated Pichia ciferrii cell according to claim 10wherein said serine palmitoyl transferase is encoded by SEQ ID No 13and/or SEQ ID No
 15. 12. An isolated Pichia ciferrii cell according toclaim 5, wherein said enzyme E₂ is a sphinganine C4-hydroxylase.
 13. Anisolated Pichia ceferrii cell according to claim 12, wherein saidsphinganine C4-hydroxylase is encoded by SEQ ID No
 17. 14. The methodaccording to claim 7, wherein said foreign DNA is DNA coding for aselection marker gene.
 15. The method according to claim 7, wherein saidreplacement of parts of the gene with foreign DNA is of the promoterregion.